Various attempts have been made to purify PAI-1, mutants of PAI-1 and fragments thereof. However, these attempts have required multiple chromatographic steps and/or alteration of the protein, e.g. insertion of a 6 histidine tag at the n-terminus, to achieve efficient purification.
Plasminogen activators (PAs) are specific serine proteinases that activate the proenzyme plasminogen, by cleavage of a single Arg-Val peptide bond, to the enzyme plasmin (Saksela O, Biochim Biophys Acta (1985) 823:35–65). Two plasminogen activators are found in mammals, tissue-type PA (tPA) and urokinase-type PA (uPA) (Saksela O et al, Annu Rev Cell Biol (1988) 4:93–126). These enzymes are thought to critically influence many biological processes, including vascular fibrinolysis (Bachmann E, Thromb Haemost (1987) 10:227–265), ovulation (Hsuch A J W et al, In: Haseltine FP et al, eds, “Meiotic Inhibition: Molecular Control of Meiosis” New York: Liss 1988:227–258), inflammation (Pollanen J et al., Adv Cancer Res (1991) 57:273–328), tumor metastasis (Dano K et al., Adv Cancer Res (1985) 44:139–266), angiogenesis (Moscatelli D et al., Biochim Biophys Acta (1988) 948:67–85), and tissue remodeling (Saksela, Annu Rev Cell Biol (1988) 4:93–126).
The regulation of PAs is a complex process controlled on many levels. The synthesis and release of PAs are governed by various hormones, growth factors, and cytokines (Saksela, Annu Rev Cell Biol (1988) 4:93–126; Dano et al., Adv Cancer Res (1985) 44:139–266). Following secretion, PA activity can be regulated both positively and negatively by a number of specific protein-protein interactions. Activity can be enhanced or concentrated by interactions with fibrin (Hoylaerts M et al., J Biol Chem (1982) 257:2912–2919), the uPA receptor (uPAR) (Ellis V et al., Semin Thromb Hemost (1991) 17:194–200), the tPA receptor (tPAR) (Hajjar K A et al., J Biol Chem (1990) 265:2908–2916), or the plasminogen receptor (Plow E F et al., Thromb Haemost (1991) 66:32–36).
PA activity can be down regulated by specific PA inhibitors (PAIs) (Lawrence, D. A et al., In: Molecular Biology of Thrombosis and Hemostasis, Roberts, H. R. et al., (Eds.), Marcel Dekker Inc., New York, chapter 25, pp. 517–543 (1995). The PAIs have become recognized as critical regulators of the PA system. Four kinetically relevant PAIs are currently recognized: PAI type 1 (PAI-1), initially described as the endothelial cell PAI; PAI type 2 (PAI-2), also referred to as placental PAI; PAI type 3 (PAI-3), also known as activated protein C (APC) inhibitor and proteinase nexin 1 (PN-1), also called glia-derived neurite-promoting factor.
PAI-1 is considered one of the principal regulators of the PA system. It is a single chain glycoprotein with a molecular weight of 50 kDa (Van Mourik J A et al., J Biol Chem (1984) 259:14914–14921) and is the most efficient inhibitor known of the single- and two-chain forms of tPA and of uPA (Table 1) (Lawrence D et al., Eur J Biochem (1989) 186:523–533). PAI-1 also inhibits plasmin and trypsin (Hekman C M et al., Biochemistry (1988) 27:2911–2918) and also inhibits thrombin and activated protein C, though with much lower efficiency.
PAI-1 exists in an active form as it is produced by cells and secreted into the culture medium and an inactive or latent form that accumulates in the culture medium over time (Hekman C M et al., J Biol Chem (1985) 260:11581–11587; Levin E G et al., Blood (1987) 70:1090–1098). The active form spontaneously converts to the latent form with a half-life of about 1 h at 37° C. (Lawrence et al., Eur J Biochem (1985) 186:526–533; Hekman et al., Biochemistry (1988) 27:2911–2918; Levin E G et al. (1987) Blood 70:1090–1098).
The latent form can be converted into the active form by treatment with denaturants, negatively charged phospholipids or Vn (Hekman et al. Biochemistry (1988) 27:2911–2918; Wun T-C et al, J Biol Chem (1989) 264:7862–7868). The reversible interconversion between the active and latent structures, presumably due to a conformational change, is a unique feature of PAI-1 as compared to other serpins. The latent form appears to be more energetically favored.
Increased levels of circulating PAI-1 are associated with thrombotic disease, including myocardial infarction and deep vein thrombosis (Juhan-Vague I et al., Thromb Res (1984) 33:523–530; Hamsten A et al., N Engl J Med (1985) 313:1557–1563; Wiman B et al., J Lab Clin Med (1985) 105:265–270; Paramo J A et al., BMJ (1985) 291:573–574; Nilsson I M et al., BMJ (1985) 290:1453–1456; Aznar J et al., Br Heart J (1988) 59:535–541; Angles-Cano E et al., J Lab Clin Med (1993) 121-:646–653). Reduced postoperative fibrinolytic activity has been correlated with increased PAI-1 activity immediately following surgery (Kluft C et al., Scand J Clin Lab Invest (1985) 45:605–610), apparently mediated by a plasma factor that stimulates PAI-1 production and secretion from vascular ECs (Kassis J et al., Blood (1992) 80:1758–1764). Consistent with these observations, the overproduction of PAI-1 in transgenic mice results in venous thrombosis primarily in the extremities (Erickson L A et al., Nature (1990) 346:74–76). In contrast, a prospective study found no correlation between PAI-1 levels and vascular disease (Ridker P M et al., Circulation (1992) 85:1822–1827).
There is evidence that the PA system with its key components uPA, its cell surface receptor uPAR and its inhibitor, PAI-1 plays a key role in tumor invasion and metastasis. Structure based design has led to the generation of mutant PAIs which are very selective and are useful for controlling tumor invasion and metastasis. Wilex et al., 2001, Expert Opin. Biol. Ther. 4:693.
U.S. Pat. No. 6,103,498 (“the '498 patent”) of Lawrence et al. (incorporated herein by reference) describes mutants of PAI-1 that have been shown to interact with and inhibit elastase and to inhibit vitronectin-associated cell migration. Because of the role of elastase in emphysema, cystic fibrosis (CF) and in acute respiratory distress syndrome (ARDS) in both adults and infants, the '498 patent indicates that the disclosed mutants of PAI-1 are useful for treating these diseases and any other diseases associated with the pathogenic activation of elastase. Lawrence et al. (Biochemistry (1994) 33:3643; incorporated herein by reference) also have generated mutants of PAI-1 which have a shorter half life when compared to wild-type PAI-1 but retain wild-type activity. In addition, Sherman et al. (J. Biol. Chem. (1992) 267:7588–7595; incorporated herein by reference) have generated mutants of PAI-1 to investigate the role of the reactive center residues of PAI-1 and showed that some of the mutants were inactive, some had a greater preference for uPA and some had a greater preference for tPA than wild-type PAI-1.
U.S. Pat. No. 5,866,413 of Sambrook et al. (“the '413 patent”; incorporated herein by reference) describes PAI-1 mutants which are capable of binding to mutant serine proteases which have become resistant to inhibition by wild-type PAI-1. These mutant PAI-1s may be useful for treating diseases linked to the mutant serine proteases. The '413 patent indicates that the PAI-1 mutants may be useful for inhibiting a mutated t-PA in a patient treated for a thrombotic disorder during an invasive procedure.
The importance of PAI-1 and fragments and mutants of PAI-1 as therapeutic compounds is increasingly being acknowledged. It is therefore desirable to purify PAI-1, PAI-1 mutants and fragments thereof to high purity and in large amounts for therapeutic use. Several purification schemes have been described, many of which require several steps to achieve high purity. It is often the case that purification schemes which require several steps can compromise the activity of the protein that is being purified.
Lindahl and Wiman (Biochim Biophys Acta (1989) 23:994) describe a method for purifying high and low molecular weight PAI-1 from fibrosarcoma cell-line HT 1080 which includes an affinity chromatography step (heparin-sepharose), a gel filtration step (Sephadex G-150) and then another affinity chromatography step (resin comprising a monoclonal antibody towards PAI-1).
Wun et al. (J. Biol. Chem. (1989) 264:7862–7868) describe an affinity purification of PAI-1 using a resin including an immobilized anhydrourokinase. PAI-1 was purified from both Human Hep G2 hepatoma cells and HT 1080 cells. The PAI-1 purified from the Hep G2 cells was bound to Vn and it was found to be four fold more active than PAI-1 purified from the HT 1080 cells which was not bound to Vn.
Alessi et al. (Eur. J. Biochem (1988) 175:531–540) describe a three step procedure for purifying both endogenous and recombinant PAI-1 which includes a zinc-chelate-Sepharose step, a gel filtration step using Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody. The recovery was about 20%. The gel filtration step is required to separate an active high molecular weight fraction of PAI-1 (which is in the void volume of the column) from an inactive low molecular weight fraction of PAI-1.
In addition, PAI-1 has been purified through the use of an n-terminal six histidine (His6) tag. See Arroya De Prada et al., Eur J Biochem 269:184–192 (2002). N-terminal 6 histidine tags are routinely added to proteins in order to facilitate purification. Arroya de Prada et al. engineered ten different PAI-1 variants, as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli to include a N-terminal His6 tag and purified by affinity chromatography using a nickel chelating column. Even in the presence of the His6 tag, after a single purification step using the nickel chelating column, the protein was only moderately purified. A second purification step using the nickel chelating column was necessary to achieve >95% purity.
Reilly et al. describe a purification scheme for PAI-1 which includes utilization of sequential anion exchange and cation exchange chromatography on Q-sepharose and S-sepharose columns, with a resultant specific activity of 250,000 units/mg based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. See Reilly et al., J. Biol. Chem. 265:9570–9574 (1990). A one step purification scheme for recombinant PAI-1 from E. coli is described by Sancho et al. using an ion exchange resin, CM-50 Sephadex, with a resultant activity of 132,000 units/ml or 14,666 units/mg.
Although other methods of purifying PAI-1 have been described, these methods are either cumbersome and involve multiple steps or require alteration of the protein to include a purification tag. It is desirable to provide a simple purification method for PAI-1 which allows for efficient purification while not requiring the alteration of the protein for the purpose of purification.